This chapter begins by comparing the organization of DNA in bacteria and eukaryotes. The possibility that similar mechanisms are utilized by both eukaryotes and prokaryotes to separate or disentangle sister chromatids or daughter strands is outlined. It is suggested that transcriptional activity and resulting RNA-protein (hnRNP) particles may play a role in DNA strand separation in eukaryotic cells, just as coupled transcription-translation does in prokaryotes. Two characteristics of a bacterial cell, such as Escherichia coli, may contribute to the difficulty in understanding its mechanism of genome segregation: first, the occurrence of DNA synthesis throughout the whole cell cycle during rapid growth, and second, the lack of a unique centromere sequence, which is characteristic of the eukaryotic chromosome. In vitro studies have shown that phenomena like phase separation, monomolecular collapse, and intermolecular aggregation of the DNA into a condensed state can be induced by high concentrations of proteins (macromolecular crowded solution) and ions. Visualization of RNA transcription and DNA replication has shown that these two processes occur in hundreds of different domains scattered throughout the S-phase nucleus. The chapter discusses possible mechanisms used in bacteria for movement of DNA and for nucleoid segregation. It also focuses on separation of daughter strands in bacteria, and separation of sister chromatids in eukaryotes. The genomic organization may help in chromosome segregation, the expression of genes fulfilling a role in the fundamental process of daughter strand separation.

(A) Comparison of the size of the spindle apparatus in a HeLa cell and the size of an E. coli cell grown in glucose minimal medium. (B) The displacement of DNA between the sister chromatids in a metaphase chromosome is comparable to that of the segregated nucleoids in E. coli. The spots indicate tubulin or, in E. coli, the tubulin-like FtsZ proteins, which form a ring structure prior to division (40, 53).

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FIGURE 1

(A) Comparison of the size of the spindle apparatus in a HeLa cell and the size of an E. coli cell grown in glucose minimal medium. (B) The displacement of DNA between the sister chromatids in a metaphase chromosome is comparable to that of the segregated nucleoids in E. coli. The spots indicate tubulin or, in E. coli, the tubulin-like FtsZ proteins, which form a ring structure prior to division (40, 53).

The fundamental difference between the modes of segregation in prokaryotes and eukaryotes becomes evident in the case of multichromosomal cells. (A) In a schematic haploid cell, the chromosome, depicted as a solid rod, is semiconservatively replicated, generating two daughter chromosomes, depicted as a solid rod and a dashed rod. In the prokaryotic cell, these chromosomes are segregated into the daughter cells as indicated. Likewise, in the eukaryotic cell, they are segregated as sister chromatids pulled by spindle microtubuli to opposite poles. (B) In a multichromosomal, or diploid, cell, replication of the two chromosomes results in two pairs of chromosomes (chromatids), depicted schematically as solid and shaded rods and dashed rods. In the prokaryotic cell, each pair of daughter chromosomes is located in a prospective daughter cell (as in eukaryotic meiosis I); these daughter chromosomes become segregated only in the next cycle. This has been called hierarchical segregation by Donachie et al. (12). In the eukaryotic cell, each pair of daughter chromosomes or sister chromatids becomes segregated into different daughter cells because of the cohesion between chromatids and because of the attachment of microtubuli from opposite poles to the kinetochores. If a mutation occurs in one of the chromosomes, as indicated by the white stars, the cell becomes heterozygous. As proposed by Donachie et al. (12), hierarchical segregation does not maintain this heterozygosity whereas mitotic segregation does.

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FIGURE 2

The fundamental difference between the modes of segregation in prokaryotes and eukaryotes becomes evident in the case of multichromosomal cells. (A) In a schematic haploid cell, the chromosome, depicted as a solid rod, is semiconservatively replicated, generating two daughter chromosomes, depicted as a solid rod and a dashed rod. In the prokaryotic cell, these chromosomes are segregated into the daughter cells as indicated. Likewise, in the eukaryotic cell, they are segregated as sister chromatids pulled by spindle microtubuli to opposite poles. (B) In a multichromosomal, or diploid, cell, replication of the two chromosomes results in two pairs of chromosomes (chromatids), depicted schematically as solid and shaded rods and dashed rods. In the prokaryotic cell, each pair of daughter chromosomes is located in a prospective daughter cell (as in eukaryotic meiosis I); these daughter chromosomes become segregated only in the next cycle. This has been called hierarchical segregation by Donachie et al. (12). In the eukaryotic cell, each pair of daughter chromosomes or sister chromatids becomes segregated into different daughter cells because of the cohesion between chromatids and because of the attachment of microtubuli from opposite poles to the kinetochores. If a mutation occurs in one of the chromosomes, as indicated by the white stars, the cell becomes heterozygous. As proposed by Donachie et al. (12), hierarchical segregation does not maintain this heterozygosity whereas mitotic segregation does.

(A) Comparison of a diploid yeast cell, its nucleus, and the diameter of a HeLa cell nucleus with an E. coli cell. See Table 1 for a calculation of the DNA concentrations in these compartments. (B) Regions (200 nm wide) of a yeast nucleus and an E. coli cell. The chromatin in the yeast nucleus (left panel) is depicted as a folded 130-nm-wide thread (4) formed by folding of the 30-nm-diameter fiber (see the text). Nucleosomes are depicted as 10-nm-diameter circles. The large circles represent hnRNP particles with diameters of 20 nm (30) involved in cotranscriptional processing of pre-mRNA. The DNA in the E. coli nucleoid is drawn as plectonemic supercoils with diameters of 20 nm (see chapter 10). Ribosomes, with diameters of about 30 nm, are involved in cotranscriptional translation.

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FIGURE 3

(A) Comparison of a diploid yeast cell, its nucleus, and the diameter of a HeLa cell nucleus with an E. coli cell. See Table 1 for a calculation of the DNA concentrations in these compartments. (B) Regions (200 nm wide) of a yeast nucleus and an E. coli cell. The chromatin in the yeast nucleus (left panel) is depicted as a folded 130-nm-wide thread (4) formed by folding of the 30-nm-diameter fiber (see the text). Nucleosomes are depicted as 10-nm-diameter circles. The large circles represent hnRNP particles with diameters of 20 nm (30) involved in cotranscriptional processing of pre-mRNA. The DNA in the E. coli nucleoid is drawn as plectonemic supercoils with diameters of 20 nm (see chapter 10). Ribosomes, with diameters of about 30 nm, are involved in cotranscriptional translation.

(A) Working model of transcription-mediated segregation assuming (i) a dedicated mechanism for the initial displacement of the two replicated origins (oriC and oriC′, indicated by the round and square symbols, respectively) and (ii) the expansion of the nucleoid by the transient attachment of DNA loops through cotranscriptional and cotranslational translocation of membrane proteins, forming two membrane growth zones that cause cell elongation. Solid and shaded loops attached to the membrane represent DNA from the two replicated daughter strands. See Fig. 2 in reference 81 for a more detailed representation of this attachment complex. (B) Initial displacement of the origins could occur by the formation of a hypothetical ribosome assembly compartment formed at rRNA genes near the unique origin of replication. Through this cotranscriptional assembly the origins are pushed apart (open arrows). The movement of this initial displacement is subsequently taken over and enhanced by DNA loops pulled to the membrane during the cotranscriptional and cotranslational translocation of membrane proteins (transertion [57]). This segregation mechanism is also suitable for multifork replication. When, after reinitiation, the origins of each pair are moved apart along the cell's long axis, the subsequently replicated daughter strands will again generate loops that attach to the membrane, now forming two pairs of growth zones. (Bar, 150 nm.)

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FIGURE 4

(A) Working model of transcription-mediated segregation assuming (i) a dedicated mechanism for the initial displacement of the two replicated origins (oriC and oriC′, indicated by the round and square symbols, respectively) and (ii) the expansion of the nucleoid by the transient attachment of DNA loops through cotranscriptional and cotranslational translocation of membrane proteins, forming two membrane growth zones that cause cell elongation. Solid and shaded loops attached to the membrane represent DNA from the two replicated daughter strands. See Fig. 2 in reference 81 for a more detailed representation of this attachment complex. (B) Initial displacement of the origins could occur by the formation of a hypothetical ribosome assembly compartment formed at rRNA genes near the unique origin of replication. Through this cotranscriptional assembly the origins are pushed apart (open arrows). The movement of this initial displacement is subsequently taken over and enhanced by DNA loops pulled to the membrane during the cotranscriptional and cotranslational translocation of membrane proteins (transertion [57]). This segregation mechanism is also suitable for multifork replication. When, after reinitiation, the origins of each pair are moved apart along the cell's long axis, the subsequently replicated daughter strands will again generate loops that attach to the membrane, now forming two pairs of growth zones. (Bar, 150 nm.)

(A) Recently replicated sister chromatids, drawn as solid and shaded 30-nm-diameter fibers, are locally disentangled by topoisomerases and a pulling and pushing force exerted by transcriptional microcompartments. These are formed around one daughter strand by the activities of cotranscriptional splicing and transport of pre-mRNA. Compare this with the initial separation of replicated oriC in a bacterium (Fig. 4B). The force is directed perpendicularly to the long axis of the chromatids (open arrows). (B) Condensation of the DNA in two separated (euchromatin) regions exerts a pulling force on the chromatids (small arrows) and gives directionality for further disentanglement by topoisomerases. Transcription may separate the strands locally, allowing them to slide past each other to the regions of condensation. The cohesion between sister chromatids is shown here as the result of residual entanglements, but it could also be achieved by specific binding proteins (32) (Bar, 150 nm.)

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FIGURE 5

(A) Recently replicated sister chromatids, drawn as solid and shaded 30-nm-diameter fibers, are locally disentangled by topoisomerases and a pulling and pushing force exerted by transcriptional microcompartments. These are formed around one daughter strand by the activities of cotranscriptional splicing and transport of pre-mRNA. Compare this with the initial separation of replicated oriC in a bacterium (Fig. 4B). The force is directed perpendicularly to the long axis of the chromatids (open arrows). (B) Condensation of the DNA in two separated (euchromatin) regions exerts a pulling force on the chromatids (small arrows) and gives directionality for further disentanglement by topoisomerases. Transcription may separate the strands locally, allowing them to slide past each other to the regions of condensation. The cohesion between sister chromatids is shown here as the result of residual entanglements, but it could also be achieved by specific binding proteins (32) (Bar, 150 nm.)

51.Murphy, L. D.,, and S. B.Zimmerman. 1995. Condensation and cohesion of λ DNA in cell extracts and other media: implications for the structure and function of DNA in prokaryotes. Biophys. Chem.57:71–92.